1. Field of the invention
This invention relates to a medium for electrophoresis, and more particularly relates to a medium for electrophoresis suitably employable for analysis of biopolymers such as proteins, as well as for determination of base sequence of DNA, RNA, their fragments, and their derivatives.
2. Description of prior arts
For the analysis of biopolymers such as proteins, or for determination of base sequence of DNA or RNA, electrophoresis can be carried out in the following manner.
A membrane medium for electrophoresis prepared by coating or casting a membrane-forming material such as agar, cellulose, cellulose acetate, starch, silica gel or polyacrylamide gel over a support such as a glass plate or a transparent plastic sheet (or film) is impregnated with a buffer solution; on the medium is applied a substance to be analyzed (sample); the applied sample is developed (or resolved) on or in the medium by applying a voltage between the both ends of the support, and then the developed substance is dyed thereon; and then the dyed sample is measured on the optical density to quantitatively determine the developed components of the sample.
Details of the electrophoresis and medium therefor are given in "Experimental Text for Electrophoresis (5th revision)" edited by Electrophoresis Society of Japan (Bunkodo, 1975), "Modern Electrophoresis" edited by Aoki & Nagai (Hirokawa Shoten, 1973), etc.
Recently, the electrophoresis has been frequently employed to analyze substances originating from a living body; for instance, the analyses of proteins originating from a living body are generally performed in the course of biochemical analysis for diagnosis. The determinations of base sequences of DNA or RNA are also performed in the course of the study in the genetic engineering technology.
As the membrane or sheet for electrophoresis, a filter paper was previously employed, but recently an agarose membrane or a polyacrylamide gel membrane (or medium) has been employed from the viewpoints of their advantageous properties. Particularly, the polyacrylamide gel membrane showing a molecular sieve function is widely employed recently. More particularly, in the method for determination of base sequence of DNA, RNA, their fragments, and their derivatives according to the post-label method, a procedure of slab electrophoresis using a polyacrylamide gel membrane has become essential.
The polyacrylamide gel membrane is prepared by crosslinking polymerization of a monomer such as acrylamide and a two-functional crosslinking agent such as N,N'-methylenebisacrylamide under an oxygen-free condition in the presence of water and a polymerization catalyst.
In the course of the preparation of the polyacrylamide gel membrane, a modifier such as an anionic surfactant, urea or formamide may be incorporated into the membrane. When only a small amount of the modifier is required for the preparation of the gel membrane for protein analysis, the modifier can be incorporated into the membrane by applying an aqueous modifier solution onto the wet gel membrane or immersing the gel membrane in an aqueous modifier solution.
Since the polymerization reaction for the preparation of polyacrylamide is a radical crosslinking polymerization as described above, the polymerization can be easily inhibited by the presence of oxygen. Therefore, the gel membrane should be prepared in the absence of oxygen. For this reason, a polyacrylamide gel membrane is generally prepared by a process involving: introducing an aqueous solution (gel-forming solution or gel solution) containing acrylamide, a crosslinking agent and a polymerization catalyst into a cell formed between two glass plates with a certain clearance (e.g., 0.3-1 mm); sealing the gel-forming solution from oxygen; and causing the crosslinking polymerization to prepare the desired gel membrane.
The polyacrylamide gel membrane prepared as above is employed for electrophoresis. For example, the electrophoresis for determination of base sequence of DNA is performed in the manner such as described below.
The polyacrylamide gel membrane is vertically placed in the form of being sandwiched between the glass plates, and in the first place a pre-electrophoresis procedure is carried out. Then, a certain amount of a sample (e.g., .sup.32 P-labeled DNA cleaved by Maxam-Gilbert method) is introduced into sample slots provided on the membrane, and electrophoresis is carried out. After the electrophoresis is carried out for a certain period of time (e.g., approx. 6-12 hours), one glass plate is removed carefully. Then, the exposed gel membrane is covered with a polymer film such as a poly(vinylidene chloride) film and subjected to the autoradiographic process. The autoradiographic process is carried out by the following procedures: A radiographic film and an intensifying screen are superposed in layers on the film covering the gel membrane, whereby exposing the radiographic film to the gel membrane at a low temperature (e.g., -80.degree. C.) for a certain period of time (e.g., approx. 10-20 hours). After the exposing procedure, the radiographic film is developed, and the resolved pattern reproduced on the film is studied for determination of the base sequence of DNA, etc.
The above-described conventional polyacrylamide gel membrane is easily breakable, so that it is practically impossible to establish a system of preparing the polyacrylamide gel membrane in a mass scale and supplying technicians of electrophoresis with it in response to their requests. But recently, as more frequently the electrophoresis is employed to analyze substances originating from a living body, demand for polyacrylamide gel membrane has been sharply increased. Thus it is desired to establish the above-described mass-production system.
One of the available methods for preparing a polyacrylamide gel membrane in a mass scale comprises conveying a continuous plastic film at a constant rate, coating thereof a gel forming solution, and radically polymerizing the coated layer in an inert atmosphere (e.g., nitrogen) to form a cross-linked membrane. However in order to carry out the said method effectively, the rate of forming the gel from the said gel forming solution should be sufficiently high. But, the conventional crosslinking agent which has been used for preparing the polyacrylamide gel membrane hardly meet the above requirement.